Evaluating polyphenolic antioxidant protection of mitochondrial DNA in a variety of human skin models

Abstract

Compromised skin barrier function negatively impacts up to 50% of our global population. Skin areas with compromised barrier integrity are affected more by environmental triggers such as sun exposure and environmental pollution which induce cellular oxidative stress, damage skin integrity and promote skin aging. At a sub-cellular level there are accumulative strand breaks to mitochondrial DNA (mtDNA). Limited repair mechanisms and proximity to the site of superoxide generation, make mtDNA a biomarker of oxidative damage in multiple human cell and tissue types. This project optimised a long-range qPCR method to compare relative mtDNA damage between samples. An application of this methodology tested commercial antioxidants (AOXs) tetrahydrocurcumin (THC) and pterostilbene (PTERO) for their capacity to protect skin mtDNA from sun exposure in a wide range of human skin models; primary monocultures, skin equivalents, and whole skin ex-plants. The assay was also used to detect mtDNA damage between donor-matched basal cell carcinoma (BCC) and non-BCC facial skin. In skin monolayer experiments, the mtDNA of foreskin-derived adult keratinocytes or differentiated keratinocytes was not protected by THC or PTERO. However the mtDNA of foreskin-derived adult reticular fibroblasts was protected by THC (but not PTERO), with greatest protection provided by a THC + PTERO combination. Various technical protocols were developed in this thesis, including an in-house full thickness human skin equivalent using CELLnTEC medium. Skin equivalents have donor-matched layers and no scaffold material, so fibroblasts secrete and maintain their own dermal extracellular matrix. A majority of the skin equivalents were formed with foreskin-derived adult keratinocytes and reticular fibroblasts, but some trials were performed using the same cell types derived from adult facial skin. In-house (CELLnTEC) and commercial (Labskin™) skin equivalents, foreskin and facial ex-plant skin were also tested for mtDNA protection by THC/PTERO/combination within topically applied Physiogel™ base formulations. As the epidermis can be manually removed from Labskin™, the qPCR assay detected a high 64-fold range of mtDNA damage in this epidermis and in-house (CELLnTEC) skin equivalents. However, the qPCR assay sensitivity was reduced in thicker skin models such as whole Labskin™, foreskin and facial ex-plant skin. As such it was difficult to determine conclusive results from formulation testing in these models using the mtDNA qPCR assay. It is anticipated that the mtDNA assay and human skin equivalent arising from this thesis will have wide-ranging applications. The data pertaining to THC/PTERO is indicative that these AOXs could enhance the GSK commercial Physiogel™ skin range designed for skin that is particularly reactive to environmental stressors such as sunlight.